Comparative Physiology @ Guelph
Culture of mouse keratinocytes and feeder cells
Culture conditions: 32◦C, 5% CO2
Mouse keratinocytes from C57Bl6 strain.
Keratinocytes grow optimally on feeder cells (e.g. 3T3J2 fibroblasts) or on collagen I-coated surfaces.
Thawing and plating cells
- Thaw 1 freezing vial containing 2x106 cells (37°C water bath).
- Transfer cells into 15 ml plastic tube containing 9 ml of pre-warmed medium and spin for 5 min at 1.000 rpm (at room temperature).
- Re-suspend in 3 ml of FDA medium and plate on collagen-coated (5 μg/cm2) culture dish (3 cm diameter).
- Change medium next day.
- To maintain their proliferative potential, Ca2+ must be depleted to 50 μM in the serum and must be absent from all solutions (e.g. PBS, DMEM etc…).
- Passage cells only when they are confluent (density 5 to 6x104 cells/cm2).
- Typical dilution is 1:2 (1:3 is the maximum).
- Trypsinize cells with 0.1% trypsin/0.02% EDTA (5 min at 37°C) until cells detach.
- Pellet the cells (1000 rpm for 4 minutes, RT).
- Re-suspend cells in fresh medium and re-plate onto 2 dishes.
- Replace medium every 2-3 days.
2x106 cells/mL per vial (90 % FBS low Ca2+ and 10% DMSO).
Induction of terminal differentiation by addition of Ca2+
- The next day after cells were seeded, change the medium from normal FAD media to FAD+ Medium containing 1.2 mM CaCl2.
- Experiments should be performed 3 days after plating and 48-50h after the calcium shift.
NB: Experiments in low calcium conditions can be performed on the second day after plating.
Culture of keratinocyte Feeder cells
- MEFs (3T3J2) fibroblasts.
- SNLP (3T3J2) fibroblasts.
- Feeder cells grow in FDA medium, without insulin, cholera toxin and hydrocortisol.
- Typical passage 1:6 (1:10 is maximum). Use 0.05% Trypsin/EDTA.
- Before using them as feeders, cells must be inactivated by mitomycin C-treatment.
- Add 10 μg/ml mitomycin C to a confluent culture of feeder cells and place in incubator for 2-3 h.
- Wash dishes 5 times with PBS and 1 time with medium.
- Freeze cells (106 cells/ml).
- Before using them, test they no longer divide for 4-5 days. Only use after this test has been performed successfully.
- Plate feeders (50.000 cells /cm2 = confluence) on collagen I-coated dishes 2h before plating keratinocytes, or together.
from Thomas Magin posted by Oualid Haddad 2013